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Human endothelium in cell culture
JOURNAL OF ATHEROSCLEROSISRESEARCH
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D. G. FRYER, G. BIRNBAUM AND C. N. LUTTRELL t Department of 3~Iedicine, Division of Neurology, University of Washington School of Medicine, Seattle, Wash. (U.S.A.) (Received February 2rid, 1965)
INTRODUCTION Tissue culture of abnormal human endothelial cells and of human endothelium mixed with cells of nonendothelial origin has been reported on several occasions. MURRAY AND STOUT1, CAMERON AND CHAMBERS2 and COMAN3 reported cultures of human tumors presumed to be of endothelial origin. WOODARDAND POMERAT 4 grew capillaries and extravascalar cells from fragments of human rib marrow pulp and LAZZARINI5 cultured intimal cells from human aorta. Relatively pure calture of normal rabbit endothelium has been claimed by SHIBUYA6, KHLOPIN AND CHISTOVA7 and by POMERATAND SLICK8. It is to MARUYAMA 9, however, that we owe the only descliption of successfal pure culture of normal human endothelium. MARUYAMAused human umbilical vein and obtained endothelial cells by perfusion with trypsin. Our belief that cell cultures derived in this way might prove of value in studies related to vascular physiology led us to repeat and extend MARUYAMA'Sexperiments with the purpose of obtaining a clearer idea of the characteAstics of the cellular elements involved. The additional information obtained in our work is presented in this communication. METHODAND MATERIAL The method of obtaining human endothelial cells from umbilical cords was adapted from that described by MARUYAMA9. Strict aseptic technique was observed in all handling of the cord. The cord was severed from the placenta and placed in a sterile container within a few minutes of birth. When endothelial cells were to be obtained from the umbilical vein, the container could be stored in a refrigerator at 4~ The specimen appeared to suffer no damage if left cold for 3-4 hours before perfusion. However, when endothelial cultures were prepared from an umbilical artery, it was necessaly to keep the cord at 37~ until preparations fol pe, fusion were made. * Dr. Luttrell died on May 16, 1964. J. Atheroscler. Res., 6 (1966) 151-163
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If the cord was allowed to cool, the arteries constricted and subsequent perfusion was impossible. Before perfusion the cord was wiped clean of blood, and any part which had been damaged by the use of clamps during delivery was cut off and discarded. The vessel chosen was picked up b y its cut edge with fine-toothed forceps and a blunt, 15 gauge needle about 1.5 in. long was inserted into it and kept in place by means of an artery forceps applied across the entire cord. The vessel was slowly perfused through the needle with 100 ml of calcium and magnesium-free saline (GKN) to wash out all the blood. The cord was then held vertically, and the vessel allowed to drain. An artery forceps was applied across the cord at the end opposite the needle, and 10-15 ml of 0.25 ~ trypsin solution in G K N were syringed into the vessel, and the needle closed with a rubber stopper. The entire cord was placed in a petri dish, covered with H a n k ' s solution and incubated at 37~ for 45 minutes. Following incubation the cord was suspended vertically with the needle down, the stopper removed from the needle, and the contained suspension of endothelial cells drained into a beaker containing 20 m] of growth medium. The contents of the beaker were then centrifuged at 500 r.p.m, for 15 minutes, and the supernatant fluid decanted leaving a " b u t t o n " of cells. The " b u t t o n " was dispersed in growth medium by repeated pipetting, and 1 ml of cell suspension placed in each Leighton tube. The yield from the vein was usually sufficient to provide fairly continuous monolayei s on 5-6, 11 • 4 m m cover; slips in Leighton tubes, while one altery provided enough cells for 2-4 such monolayers. To obtain confluent monolayers it was necessaiy to use concentrations of 400,000-600,000 cells/ml. Living cultures on coverslips in Leighton tubes were observed with an inverted microscope. Cultures were also grown in Rose chambers and by the double coverslip method in which a drop of the medium containing suspended cells was held between two 43 • 50 m m coverslips separated by a metal slide 0.5 m m thick with a 2 cm hole in its center. Rose chambers and metal slides permitted the obserVation of living cells b y phase contrast microscopy. Most of the cells were grown directly on the glass coverslip surface, but reconstituted rat-tail collagen (prepared by the method described b y EHRMANN AND GEY 10) was also used as a substrate on the coverslip in a few experiments. The culture media were obtained from Microbiological Associates. In most experiments the growth medium used was 199 with 20 ~ calf serum (inactivated at 56~ for 30 minutes) containing 100 units/ml of penicillin, 100 #g/ml streptomycin and 50/~g/ml micostatin. The p H was adjusted to 7.4 with 10 % sodium bicarbonate. In some experiments human cord serum was substituted fol fetal calf serum. In order to examine the appearance of the umbilical vessels before and after trypsinization, segments of cords were embedded in paraffin, sectioned and stained with Verhoeff's elastic stain. To determine whether the internal elastic lamina was damaged in the process of removing the endothelium, some cords were sectioned at each cm, including the end regions where forceps had been applied. Fibroblasts were grown from umbilical cord and from foreskins of circumcised male infants. A small portion of umbilical cord or foreskin was cut into pieces about 1 m m diameter with two scalpels, 20-30 such pieces were transferred to the fiat surface j . Atherosder. Res., 6 (1966) 151-163
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of a 100 ml medicine bottle, and sufficient growth medium was added to moisten the explants. After incubation at 37~ for 1-2 days, most of the pieces were firmly adherent to tile glass, and further medium was added to cover the explants. Fibroblasts grew out from many of these pieces within 2 weeks, and by 4 weeks a monolayer of fibroblasts covered several square centimeters of the glass surface. This monolayer was harvested with a solution composed of 0.05 % trypsin and 0.2 ~ verseue in tris buffer at pH 7.4 and transferred to coverslips in Leighton tubes. Endothelial and fibroblast monolayers were usually examined after fixation in 10 % formalin in Ringer-Locke solution and staining with DELAIqELD'S hematoxylin and eosin. For special purposes Sudan IV, periodic acid-Schiff, and benzidine stains were used. In some specimens the intercellular "cement" was stained black with silver nitrate befole fixation. This was done by covering the coverslip successively with 0.4 ~ silver nitrate (2 minutes), 5 ~ glucose (3 changes), a solution containing 3 ~ cobalt bromide and 1 ~ ammonium bromide (1 minute), and finally, 5 ~o glucose (3 changes). In order to identify nuclei that were actively synthesizing DNA an autoradiographic technique was employed. The cell cultures were exposed for half an hour to culture medium containing 1/~C/ml of tritiated thymidine. The monolayers were then fixed in Ringer-Locke-formalin, dehydrated in absolute alcohol and allowed to dry for 12 hours. They were then coated with NTB-3 emulsion (Eastman Kodak), exposed in a light-tight box for 2 days, developed, and stained through the fixed emulsion with MACNEAL'S tetrachrome stain 1I. When this technique was used to compare endothelial cells with fibroblasts, the two cell types were subcultured onto fresh coverslips and treated with 3H thymidine and fixed at identical intervals after subculture. In some experiments pbagocytosis of carbon particles (India ink) by cells in culture was studied using a standard suspension made by adding 2 ml of Higgin's India ink to 100 ml of the growth medium. Phagocytosis was also studied by exposing the culture to growth medium containing human blood cells, chicken blood cells, sheep blood ceils, endothelial cells previously labelled with India ink, and heat-killed bacteria (Bacillus subtilis or Staphylococcus epidermidis). RESULTS
Venous endothelium Histologic sections of cords stained by Verhoeff's elastic stain showed that most of the endothelial cells had been detached from the trypsinized vein leaving the underlying internal elastic lamina intact. Sections of untrypsinized vein showed 51o cells lying between the endothelial Iayer and the internal elastic lamina (Figs. 1 and 2). When the cell suspensions were allowed to settle over a coverslip, the initially spherical cells began to flatten out on the glass surface within 3 hours. As the cells flattened they spread to cover a larger area, and by 12-24 hours the spreading process was complete. After this time any parts of the glass surface not covered by the cells remained uncovered. Although a slight shift in the position of individual cells occurred J. Atheroscler. Res., 6 (1966) 151-163
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after the first 24 hours, the m o n o l a y e r did n o t spread b y replication a n d m i g r a t i o n as was seen in fibroblast cultures. The flattened endothelial cells formed a sheet with each cell a b u t t i n g against the edge of neighboring cells. I n fixed p r e p a r a t i o n s there w~s often a narrow space between a d j a c e n t cells. The cells were polygonal or slightly elongated, a n d there was a r e m a r k a b l e u n i f o r m i t y in size of b o t h cell bodies a n d nuclei (Fig. 3). The cells were 40-50 # in their longest axis, a n d the nuclei 20 • 10/~ in their greatest a n d least diameters. The nucleus u s u a l l y c o n t a i n e d 1 or 2 nucleoli and very seldom more t h a n 3. Some cells c o n t a i n e d scattered sudanophilic droplets in the cytoplasm. The n u m b e r of cells with this appearance increased with the age of the culture. Silver s t a i n i n g gave black " c e m e n t lines" with endothelial cells grown either on collagen or on glass (Fig. 5). W i t h h e m a t o x y l i n staining m a n y cells showed dark, straight or slightly curved, parallel filaments (tonofibrils) in their cytoplasm. The m i t o c h o n d r i a were filamentous a n d were best seen b y phase c o n t r a s t microscopy. A few endothelial cultures were m a i n t a i n e d for 6 weeks, b u t more c o m m o n l y the cells became irregular in shape a n d detached from the s u b s t r a t u m after 2 - 5 weeks. I t was n o t possible to m a i n t a i n t h e m b y s u b c u l t u r e when glown in m e d i u m e~riched with either bovine fetal or h u m a n cord serum. W h e n cell counts were made before a n d after coverslip culture, no increase in cell n u m b e r was detected. Mitoses were occasionally seen b u t were e x t r e m e l y rare (Fig. 4). W h e n I n d i a ink suspension was added to the endothelial cultures, black granules appeared in their cytoplasm after a few hours a n d c o n t i n u e d to a c c u m u l a t e for several days (Fig. 6). Most of the granules congregated in the region m i d w a y b e t w e e n the nucleus and the edge of the cell leaving the i m m e d i a t e perinuclear a n d circumferential regions clear (Fig. 7).
Fig. 1. Cross section through wall of umbilical vein before trypsinization. The internal elastic lamina is clearly seen; and where it is most plicated, the endothelial cells assume columnar form. Verhoeff's elastic stain; • 188. Fig. 2. Cross section through wall of umbilical vein after trypsinization, Most of the endothelial cells have been detached, and the internal elastic lamina remains as a barrier preserving the integrity of subendothelial tissues. Verhoeff's elastic stain; • 406. Fig. 3. Umbilical vein endothelial cell culture monolayer. Cells are fairly uniform in shape and size. Many have cytoplasmic inclusions. Hematoxylin and eosin; • 188. Fig. 4. Mitotic figure in endothelial cell culture. Hematoxylin and eosin; • 714. Fig. 5. Endothelial cell culture on glass coverslip surface stained with silver nitrate and subsequently with hematoxylin and eosin. A black "cement line" surrounds many of the cells, x 714. Fig. 6. Endothelial culture after incubation for three hours with growth medium containing India ink suspension. A spindle-shaped "atypical" cell (macrophage) is identified by black granules in the cytoplasm. Hematoxylin and eosin; • 406. Fig. 7. A rounded macrophage in the same culture as shown in Fig. 6. The ink granules are concentrated near the nucleus in the center of the cell. Hematoxylin and eosin; x 406. Fig. 8. Same culture as Figs. 6 and 7 showing a macrophage with morphologie appearance intermediate between spindle-shaped and rounded forms. Hematoxylin and eosin; • 714. Fig. 9. "Typical" endothelial cells with black granules after 24 hours exposure to India ink. Hematoxylin and eosin; X 406. Fig. 10. Fibroblast culture after same exposure to India ink as the endothelial cells in Fig. 9. The cytoplasm contains few ink granules. Hematoxylin and eosin; • 406.
j . Atheroscler. Res., 6 (1966) 151-163
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j . Atheroscler. Res., 6 (1966) 151-163
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D.G. FRYER, G. BIRNBAUM, C. N. LUTTRELL There were a n u m b e r of " a t y p i c a l " cells wich t o o k up the ink v e r y r a p i d l y a n d
within 3 - 6 hours b e c a m e intensely black (Figs. 9 and 10). Many d e v e l o p e d large, clear vacuoles and b e c a m e d e t a c h e d from the coverslip. This d a m a g e could be p r e v e n t e d by limiting the t i m e of exposure to India ink to 3 hours. The " a t y p i c a l " cells were either circular or were elongated a n d spindle-shaped, often with e x p a n d e d fan-like bulbs at one or b o t h ends of the cell. The nuclei of these cells were usually slightly smaller and stained more densely t h a n the " t y p i c a l " cells, and the c y t o p l a s m had a reticular appearance. W i t h Sudan I V staining most of these cells appeared loaded w i t h fat droplets and were t h e r e b y c o n t r a s t e d with the " t y p i c a l " cells. The i d e n t i t y of the circular and e l o n g a t e d varieties of " a t y p i c a l " cells was suggested b y the presence of i n t e r m e d i a t e forms (Fig. 8) t h a t h a d all the characteristics of a circular celI b u t had a single slender process of c y t o p l a s m e x t e n d i n g from one side of the cell. In p a t c h y m o n o l a y e r s " a t y p i c a l " cells were often found in isolation some distance a p a r t from the confluent endothelial cell patches. W h e n elongated " a t y p i c a l " cells were found a m o n g " t y p i c a l " cells, t h e y a p p e a r e d to lie o v e r t h e m r a t h e r t h a n p a r t i c i p a t e in the f o r m a t i o n of the continuous sheet of cells. W h e n cultures were e x a m i n e d on a w a r m stage w i t h the phase c o n t r a s t microscope, the " a t y p i c a l " cells were seen to show c o n s t a n t changes in c o n t o u r b y the c o n t i n u a l protrusion and r e t r a c t i o n of pseudopodia. I n cultures 3 or 4 weeks old the cell bodies of m a n y of these cells were g r e a t l y enlarged, up to 200 # in diameter, a l t h o u g h the nucleus showed only a slight increase in size.
Fig. 11. This specimen was produced by labelling an endothelial culture with India ink, as shown in Fig. 9. The labelled culture was trypsinized, and the separated cells suspended in culture medium covering an unlabelled monolayer of endothelial cells. After incubation for 24 hours, many inklabelled cells were found as inclusions containing black granules. Hematoxylin and eosin; • 714. Fig. 12. Inclusions in endothelial cells after 12 hours incubation in growth medium containing human red cells. Hematoxylin and eosin; • 714. Fig. 13. Endothelial cell culture after 48 hours incubation with growth medium containing human red cells. Some endothelial cells have as many as ten included erythrocytes. Hematoxylin and eosin; • 406. Fig. 14. Endothelial cell culture after 6 hours incubation in a suspension of heat-killed B. subtilis. Hematoxylin and eosin; • 714. Fig. 15. Endothelial culture cells after incubation for 3 hours in growth medium containing heatkilled Staph. epidermidis. A macrophage is seen loaded with staphylococci. Hematoxylin and eosin; • 714. Fig. 16. Culture exposed successively to 3H thymidine for half an hour and India ink suspension for 3 hours. A macrophage is shown labelled by both aH thymidine and ink. Culture stained with 3JiACNEAL'S tetraehrome through the photographic emulsion; • 714. Fig. 17. Umbilical cord fibroblast culture in which several nuclei are labelled with ZH thymidine. Cells have overlapping and anastomosing processes that form a reticulum. Unlabelled nuclei have several darkly staining nucleoli. ~[AC~X~EAL'Stetrachrome; • 714. Fig. 18. Endothelial cell culture obtained by trypsinization of umbilical artery. Culture was exposed to India ink for 3 hours and fixed at 5 days. A small macrophage labelled with ink and a large fibroblast are shown. Hematoxylin and eosin; • 188. Fig. 19. Ten day old specimen of the same culture shown in Fig. 18. Only fibroblasts are clearly recognizable. Hematoxylin and eosin; • 188. Fig. 20. Four week old umbilical vein endothelial culture showing a very large macrophage. HematoxyIin and eosin; • 188. J. Atheroscler. Res., 6 (1966) 151-163
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J. Atheroscler. Res., 6 (1966) 151-163
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In caltures one week old about 20 ceils per 1000 wele of this "'atypical" form, but the proportion showed a slight steady increase, and in 4-week cultures there were usually about 64 per 1000. When these cells were labelled with India ink, it was found that further "atypical" cells were not formed by transformation of "typical" cells. After exposure of cultures of 3H thymidine for half an hour, only 6-9 endothelial cell nuclei per 1000 were labelled. Occasional "atypical" cells were also labelled but it was not possible to show a difference in the frequency of labelling of "typical" and "atypical" cells. In old cultures in which some cells had begun to assume irregular shapes, no labelling was found after similar exposure to 3H thymidine. Many endothelial cells in culture contained large, weakly eosinophilic, PASpositive, cytoplasmic inclusions. Often these inclusions were surrounded by a nalrow clear zone, and frequently the nucleus was indented or pushed to one side by an inclusion. When living cultures were examined by phase contrast microscopy, it was observed that not all the initially spherical cells flattened out. A few remained spherical, and when they lay in close iuxtaposition to flattened cells, they were seen in subsequently stained preparations to have become eosinophilic inclusions. In some experiments endothelial cultures were exposed to India ink until all the cells contained numerous black granules. These cells were then trypsinized and the separated cells suspended in growth medium which was added to endothelial monolayers. Inclusions containing numeroos black granules were subsequently demonstrated in the cells of these monolayers (Fig. 11). Human, chicken and sheep erythrocytes were also phagocytosed by endothelial cultures. Most of the endothelial cells acquired from 1-10 included erythrocytes within 48 hours (Figs. 12 and 13). The included blood cells were at first recognizable by their shape and size and by staining with Ralph's benzidine stain. Within a day the inclusions became fragmented and lost the staining characteristic of erythrocytes. Killed Bacillus subtilis and Staphylococcus epidermidis suspended in the growth medium were also phagocytosed (Figs. 14 and 15). "Atypical" cells showed a much greater affinity for India ink and staphylococci than "typical" cells (Fig. 16), but there was no apparent difference between the two cell types in regard to their propensity to ingest the Bacillus subtilis or cellular particles.
Arterial endothelium Histologic sections of umbilical cold showed no distinct internal elastic lamina in the mnbilical artery, and after trypsinization of the artery there was some fragmentatioll of the subendothelial tissue. The cell cultures initially differed from those derived from vein only by the presence of cells which had all the characteristics of fibroblasts. At first these fibroblasts were few in number, but within 2 weeks these cultures could be distinguished from pure fibroblast cultures only by the presence of occasional "atypical" cells that retained India ink (Fig. 18). The orderly arrangement of the endothelial cells was completely lost in the overgrowth of fibroblasts, and "typical" endothelial cells could not be recognized with certainty (Fig. 19). J. Atheroscler. Res., 6 (1966) 151-163
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Fibroblasts
No differences were detected between fibroblasts of umbilical cord and prepuce origins. Fibroblasts were irregular in shape, often greatly elongated, and sent out processes that overlapped adjacent cells forming a network with the processes of other cells. They varied greatly in size, and in large cells the nucleus often measured 50/2 in its greatest diameter. In general the nucleoli were more numerous and stained more intensely than those of endothelial cells (Fig. 17). Silver staining showed a black "cement line" similar to that seen in endothelial cultures. Tile larger fibroblasts showed darkly staining parallel filaments (tonofibrils) in their cytoplasm. Unlike endothelial cultures, fibroblasts replicated rapidly and could be readily subcultured and maintained for several months. In experiments with taitiated thymidine fibroblast nuclei took up thymidine more frequently than endothelial nuclei, and cell counts showed that 50-70 nuclei per 1000 were labelled after 30 minutes exposure to aH thymidine. Fibroblast cultures showed a very slight capacity for phagocytosis of India ink and cellular and bacterial particles.
DISCUSSION The nature and origin of a ceil cannot be determined with certainty by its morphology in culture. Since the vein internal elastic lamina was not damaged there is no doubt that the cells in the cultures derived from veins were those which lie superficial to the elastic lamina. It is on this basis, rather than morphology, that we are confident that our cultures were comprised predominantly of endothelial cells. Although endothelial cells certainly predominated, other cells may have been included such as cells which m a y occasionally lie between the elastic lamina and the endothelium, and blood cells which had become firmly attached to the endothelium. Despite these uncertainties our study sheds some light on the nature of endothelial cells, especially their relationship to fibroblasts and macrophages. There is a long-standing controversy about the relationship of endothelial cells to fibroblasts. MAXlMOWlz, in 1925, stated that in tissue culture endothelial cells grew out of the end of an explanted piece of blood vessel but soon could not be distinguished from fibroblasts; the 1957 edition of MAXlMOW AND BLOOM'S textbook 13 still maintains that endothelial cells in the body can become changed into fibroblasts. Moreover, ALTSCttUL14 in an extensive review of the literature concluded: " I t is difficult ot impossible to distinguish in tissue culture between endothelial cells and fibroblasts. Furthermore, there is the rather common belief that endothelial cells in tissue culture will turn into fibroblasts or fibroblast-like cells." However, KHLOPIN AND CHISTOVA7 have more recently stated: "rLeither in tissue culture nor in repair regeneration is endothelium ever converted into connective tissue". Pure culture of human endothelial celts from normal blood vessels has been j . Atheroscler. Res., 6 (1966) 151-163
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reported previously only by MARUYAMA9. MARUYAMAused umbilical vein as his source of ceils but was not successful in culturing fibroblasts from umbilical cord for comparison with the endothelial cells. In our investigations fibroblasts were grown from umbilical cord and from human foreskin, and comparison with endothelial cells was, therefore, possible. The fibroblasts grown from cord and foreskin were identical in appearance and behaviour and corresponded to the description of fibroblasts (mechanocytes) in culture given by WII-LMER15. MARUYAMA9 found that the intercellular "cement" stained black with silver nitrate in endothelial cultures grown on collagen but not in those grown on glass. He considered the presence of this cement line to be evidence for the endothelial origin of the ceils, tn our own experience the cement line stained equally well in cultures grown on glass and on collagen. However, we do not think these lines are especially significant as they were found also in our fibroblast cultures. ALTSCHUL14 also pointed out that this staining property cannot be considered as specifc for a single cell type. MAXlMOW1~ attached importance to the presence of tonofibrils in identifying fibroblasts. However, MARUYAMA9 iound similar "tension striae" in endothelial cells, and we also observed this appearance in both fibroblasts and endothelial cells. Nevertheless, there were definite morphological and behaviourat differences that clearly distinguished fibroblasts from endothelial cells. "Typical" endothelial cells showed little variation in size, and they spread out to form sheets; their nucleoli stained faintly and were few in number. By contrast, fibroblasts varied greatly in size, their processes overlapped and formed networks; their nucleoli were numerous and stained intensely. Further points of distinction were the greater capacity for phagocytosis in endothelial cells, and the greater rate of replication and DNA synthesis in fibroblasts as shown by the greater number of nuclei labelled with aH thymidinelT. The "atypical" endothelial cells which we noted in small numbers in our cultures of endothelium resembled fibroblasts in being spindle-shaped, and in older cultures their cell bodies grew to a size achieved b y the largest fibroblasts. However, unlike fibroblasts, they did not replicate so fast as to overgrow the "typical" endothelial cells, they did not form a reticulum of branching cells, and they had a distinctively high affinity for India ink. In disintegrating endothelial cultures the cells were often irregular in outline and elongated. These cells showed no evidence of DNA synthesis, and there is no reason to believe that they represent a conversion of either "typical" or "atypical" endothelial cells to fibroblasts. The existence of more than one cell type in endothelial cultures has been described several times. Recently KOmE et al. 18, described "fibroblasts" and another cell which " m a y be an endothelial cell". The latter cell was rounded or oval and showed ameboid movements. These authors obtained their cultules by explanting whole thickness rabbit aorta and most probably subintimal cells were present in the cultures. SH1BUYA 6, in 1931, cultured explants of rabbit endothelium. He made the following statement: "There developed after 5-6 days around the growth of the 'typical' endoJ. Athero~cler. Res., 6 (1966) 151-163
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thelial cells, some which were different from the characteristic cells. They had spindlelike or roundish bodies". This description is very applicable to our "atypical" cells. MARUYAMA9 stated: "Infrequently some cells transformed into fibroblast-like elements. For example, in certain series of cultures made from an umbilical vein, there were no fibroblast-like cells after 2-4 days in culture, but they appeared after 10-12 days. These cells were spindle-shaped with slender processes". Again, this description fits our "atypical" cells, but neither SHIBUYA6 nor MARUYAMA9 described how the spindle-shaped cells reacted with India ink. KHLOPIN AND CHISTOVA7 cultured cells from explants of rabbit vena cava endothelium and described the appearance of the culture when India ink was added. They found: "After several days the amount of India ink taken up b y the cells increases, but remains unevenly distributed between the various cells of the growth zone. Some of them contain either few granules or none, while in others there m a y be a considerable number. Gradually, the cells which are crowded in India ink granules become rounded off and disorganized". PTOKHOV19 grew endothelial cultures from explants of rabbit blood vessels and studied the phagocytic properties of cells derived in this way. Some cells became overloaded with absorbed particles and became isolated and rounded into "ball forms" with the appearance of macrophages. LAZZARINI5 described two types of cells derived from human aortic intima, one type showing a rapid uptake of colloidal carbon and in certain circumstances becoming "ring t y p e " ceils. We cannot say whether there is a resemblance between endothelial cells obtained from human umbilical artery and vein and those derived from rabbit and human adult blood vessels. The descriptions of the rounded and ameboid "endothelial" cells of KOlDE et al. Is, the "spindle-shaped and iounded cells" of GHIBUYA 6, the "fibroblast-like elements" of MAREYAMA 9, the cells "crowded with India ink" of KtfLOPIN AND CHISTOVA 7, the "isolated and rounded" cells of PTOKHOV19and the "ring type" cells of LAZZARINI5 suggest some resemblances to tile "atypical" cells we have described, but we do not presume that they are analogous. What, then, is the significance of the two distinct endothelial cell types which we have designated as "typical" and "atypical"? We found that both cell types were recognizable as soon as the culture had flattened out. If the atypical cells were damaged by prolonged exposure to India ink, they weIe not replenished by transformation of the remaining "typical" cells. The greater abundance of these cells in older cultures m a y be explained by their more rapid replication. WILLMER20 stated that in general in tissue culture, cells change morphologically to one of three types which he called mechanocytes (fibroblasts), epitheliocytes and amebocytes. Mechanocytes have a reticular arrangement, while epitneliocytes are characterized by a tendency to adhere to each other and to coverslip surfaces and to spread in a uniform sheet with each cell firmly attached to its next-door neighbors by a cement substance. Amebocytes remain detached from one another, are strongly phagocytic, store carbon particles and show ameboid motion. Using these criteria, we would classify the "typical" cells as epitheliocytes (in WILLMER'S classification) and the "atypical" as amebocytes; however, both cell types have a remarkable ability to phagocytose cellular debris. J. Atheroscler. Res., 6 (1966) 151-163
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Our cultures were composed mostly of "typical" cells which we, thelefore, regard as endothelial cells proper. WlLLMER~0 points out that in vitro monocytes and histiocytes (macrophages) behave as amebocytes, and we think that the "atypical" cells we have described are most probably macrophages that exist in close association with endothelium. I t is well established that macrophages can be cultured from blood lymphocytes and monocytes16, 21-2a, and indeed, we have cultured cells with the appearance of "atypical" cells from cord blood. However, macrophages ("atypical" cells) appeared in our cultures in the absence of erythrocytes and were, therefore, not derived from contaminating blood. We cannot exclude the possibility that lymphocytes or monocytes had become attached to the endothelium either during fetal life or after the placental circulation ceased. The conversion c)f blood macrophages to fibroblast-like cells has been described16, zs, although other authors have not observed this change even after prolonged culture of blood macrophages 22. In our cultures some macrophages became very large, but in appearance and behaviour they remained distinct from fibroblasts. The use of trypsin m a y produce chromosomal aberrations that alter metabolism and cell growth 24. However, there is no evidence for this change in short-term cultures of the kind we gave studied, and we do Itot think that the cell differences we have seen could have been caused artifactually b y trypsin treatment. CONCLUSIONS
Normal human endothelial cells derived from umbilical vein m a y be grown in cell cultule. Endothelial cell cultures derived by tr2r of umbilical arteries were initially similar to those from the vein, but a small number of fibroblasts contaminated the cultures and replicated so rapidly that the culture soon resembled a culture of pure fibroblasts. This contamination of arterial endothelial cultures by fibroblasts was associated with the absence of a distinct ivternal elastic lamina in this artery. In cultures free of fibroblasts two distinct cell types were found. Both types could be distinguished morphologically and behaviourally from fibroblasts and neither type was converted into fibroblasts. The more numerous cell type was considered to be a true endothelial cell. The less numerous ceils were thought to be macrophages but it was not determined whether they were derived Irom intimal celIs or from blood cells that adhered to endothelium. Macrophages were not formed b y metamorphosis of ordinary endothelial cells. Both cell types had a very marked ability to phagocytose cellular and bacterial particles, macrophages usually, but not always having gleater phagocytic capacity depending on the nature of the palticle being ingested. ACKNOWLEDGEMENTS
The authors are grateful to Miss NANCY BUNKER for expert technical assistance. The funds were made available for this research from the University of Washington, General Research Support Grant, P H S number F R 05132-01. j . Atherosder. Res., 6 (1966) 151-163
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SUMMARY
H u m a n endothelial cells d e r i v e d b y t r y p s i n i z a t i o n of umbilical v e i n a n d a r t e r y were grown in cell c u l tu r e a n d c o m p a r e d w i t h fibroblasts g r o w n from e x p l a n t s of umbilical cord. Tw o t y p e s of cells were recognized in endothelial cultures: endothelial cells p r o p e r an d macrophages. T h e f o r m e r replicated v e r y slowly and could n o t be m a i n t a i n e d b y s u b c u l t m e . Macrophages c o n s t i t u t e d a b o u t 5 % of the t o t a l cell population. T h e y r e p l i c a t e d slightly more r a p i d l y t h a n t h e true en d o t h el i al cells a n d were n o t d e r i v e d from these cells. N e i t h e r cell t y p e was t r a n s f o r m e d into fibroblasts. B o t h cell t y p e s h a d m a r k e d ability to p h a g o c y t o s e cellular a n d b a c t e r i a l particles, mac~ophages sometimes, b u t n o t always, h a v i n g g r e a t e r p h a g o c y t i c c a p a c i t y d ep en d i n g on the n a t u r e of t h e particle ingested. E n d o t h e l i a l cultures derived from umbilical a r t e r y initially differed from those from the vein only b y t h e presence of a small n u m b e r of fibroblasts which, in older cultures, overgrew the en d o t h el i al cells. Th e presence of fibroblasts in arterial cultures was associated w i t h t h e absence of a distinct i u t e l n a l elastic l a m i n a so t h a t s u b e n d o t h e l i a l cells were included in the culture. REFERENCES 1 M. R. MURRAYAND A. P. STOUT, Am. J. Pathol., 20 (1944) 277. 2 G. CAMERONAND R. CHAMBERS,Am. J. Cancer, 30 (1937) 115. 8 D. R. COMAN, Cancer Res., 2 (1942) 618. 4 W . C. WOODARD AND C. M. POMERAT, Anat. Rec., 117 (1953) 663. 5 A. A. LAZZARINI,Doctoral Thesis, Cornell University, 1959. e T. SHIBUYA, Kitasato Arch. Exptl. Med., 8 (1931) 68. 7 N. G. KHLOPIN AND N. M. CHISTOVA,Dokl. Akad. Nauk S S S R , 119 (1958) 217. 8 C. M. POMERATAND W. C. SLICK, Nature, 198 (1963) 859. 9 y. MARUYAMA,Z. Zellforsch., 60 (1963) 69. 10 R . L . EHRMANN AND G. O. GEY, J. Natl. Cancer Inst., 16 (1956) 1375. Xl W. J. MACNEAL,J. Am. Med. Assoc., 78 (1922) 1122. 12 A. A. MAXIMOW,Anat. Rec., 29 (1925) 369. 18 A. A. MAXIMOWAND W. BLOOM, A Textbook of Histology, 7th edition, W. B. Saunders Co., Philadelphia, 1957, p. 47. 14 R. ALTSCHUL,Endothelium, Its Development, Morphology, Function and Pathology, MacMillan Co., New York, 1954, pp. 10, 37. 15 E. N. WILLMER, Tissue Culture. The Growth and Differentiation of Normal Tissues in Artificial Media, 3rd edition, Methuen and Co., Ltd., London, 1958, p. 9. 16 A. A. MAXlMOW,Arch. Exptl. Zellforsch., 5 (1928) 169. 17 C. P. LEBLOND, B. MESSIER AND B. KOPRIWA, Lab. Invest., 8 (1959) 296. 18 R. KOIDE, O. J. POLLACKAND D. A. BURNS, J. Atheroscler. Res., 3 (1963) 32. 10 M. P. PTOKHOV, Arch. Anat. Histol. Embryol. (Leningrad), 8 (1963) 75. 20 E. N. WILLMER, Essays on Growth and Form, Oxtord University Press, Oxford, 1945, pp. 265, 272. 21 M. R. LEWIS, Am. J. Pathol., 2 (1925) 91. e2 L . BERMAN AND C. S. STULBERG, Lab. Invest., 11 (1962) 1322. 28 W. BLOOM, Handbook of Hematology, Hoeber, Inc., New York, 1938, Sec. XX, p. 1507. 24 G. BARSKI AND R . CASSINGENA, J. Natl. Cancer Inst., 30 (1963) 865. J. Atheroscler. Res., 6 (1966) 151-163
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D. G. FRYER, G. BIRNBAUM AND C. N. LUTTRELL t Department of 3~Iedicine, Division of Neurology, University of Washington School of Medicine, Seattle, Wash. (U.S.A.) (Received February 2rid, 1965)
INTRODUCTION Tissue culture of abnormal human endothelial cells and of human endothelium mixed with cells of nonendothelial origin has been reported on several occasions. MURRAY AND STOUT1, CAMERON AND CHAMBERS2 and COMAN3 reported cultures of human tumors presumed to be of endothelial origin. WOODARDAND POMERAT 4 grew capillaries and extravascalar cells from fragments of human rib marrow pulp and LAZZARINI5 cultured intimal cells from human aorta. Relatively pure calture of normal rabbit endothelium has been claimed by SHIBUYA6, KHLOPIN AND CHISTOVA7 and by POMERATAND SLICK8. It is to MARUYAMA 9, however, that we owe the only descliption of successfal pure culture of normal human endothelium. MARUYAMAused human umbilical vein and obtained endothelial cells by perfusion with trypsin. Our belief that cell cultures derived in this way might prove of value in studies related to vascular physiology led us to repeat and extend MARUYAMA'Sexperiments with the purpose of obtaining a clearer idea of the characteAstics of the cellular elements involved. The additional information obtained in our work is presented in this communication. METHODAND MATERIAL The method of obtaining human endothelial cells from umbilical cords was adapted from that described by MARUYAMA9. Strict aseptic technique was observed in all handling of the cord. The cord was severed from the placenta and placed in a sterile container within a few minutes of birth. When endothelial cells were to be obtained from the umbilical vein, the container could be stored in a refrigerator at 4~ The specimen appeared to suffer no damage if left cold for 3-4 hours before perfusion. However, when endothelial cultures were prepared from an umbilical artery, it was necessaly to keep the cord at 37~ until preparations fol pe, fusion were made. * Dr. Luttrell died on May 16, 1964. J. Atheroscler. Res., 6 (1966) 151-163
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If the cord was allowed to cool, the arteries constricted and subsequent perfusion was impossible. Before perfusion the cord was wiped clean of blood, and any part which had been damaged by the use of clamps during delivery was cut off and discarded. The vessel chosen was picked up b y its cut edge with fine-toothed forceps and a blunt, 15 gauge needle about 1.5 in. long was inserted into it and kept in place by means of an artery forceps applied across the entire cord. The vessel was slowly perfused through the needle with 100 ml of calcium and magnesium-free saline (GKN) to wash out all the blood. The cord was then held vertically, and the vessel allowed to drain. An artery forceps was applied across the cord at the end opposite the needle, and 10-15 ml of 0.25 ~ trypsin solution in G K N were syringed into the vessel, and the needle closed with a rubber stopper. The entire cord was placed in a petri dish, covered with H a n k ' s solution and incubated at 37~ for 45 minutes. Following incubation the cord was suspended vertically with the needle down, the stopper removed from the needle, and the contained suspension of endothelial cells drained into a beaker containing 20 m] of growth medium. The contents of the beaker were then centrifuged at 500 r.p.m, for 15 minutes, and the supernatant fluid decanted leaving a " b u t t o n " of cells. The " b u t t o n " was dispersed in growth medium by repeated pipetting, and 1 ml of cell suspension placed in each Leighton tube. The yield from the vein was usually sufficient to provide fairly continuous monolayei s on 5-6, 11 • 4 m m cover; slips in Leighton tubes, while one altery provided enough cells for 2-4 such monolayers. To obtain confluent monolayers it was necessaiy to use concentrations of 400,000-600,000 cells/ml. Living cultures on coverslips in Leighton tubes were observed with an inverted microscope. Cultures were also grown in Rose chambers and by the double coverslip method in which a drop of the medium containing suspended cells was held between two 43 • 50 m m coverslips separated by a metal slide 0.5 m m thick with a 2 cm hole in its center. Rose chambers and metal slides permitted the obserVation of living cells b y phase contrast microscopy. Most of the cells were grown directly on the glass coverslip surface, but reconstituted rat-tail collagen (prepared by the method described b y EHRMANN AND GEY 10) was also used as a substrate on the coverslip in a few experiments. The culture media were obtained from Microbiological Associates. In most experiments the growth medium used was 199 with 20 ~ calf serum (inactivated at 56~ for 30 minutes) containing 100 units/ml of penicillin, 100 #g/ml streptomycin and 50/~g/ml micostatin. The p H was adjusted to 7.4 with 10 % sodium bicarbonate. In some experiments human cord serum was substituted fol fetal calf serum. In order to examine the appearance of the umbilical vessels before and after trypsinization, segments of cords were embedded in paraffin, sectioned and stained with Verhoeff's elastic stain. To determine whether the internal elastic lamina was damaged in the process of removing the endothelium, some cords were sectioned at each cm, including the end regions where forceps had been applied. Fibroblasts were grown from umbilical cord and from foreskins of circumcised male infants. A small portion of umbilical cord or foreskin was cut into pieces about 1 m m diameter with two scalpels, 20-30 such pieces were transferred to the fiat surface j . Atherosder. Res., 6 (1966) 151-163
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of a 100 ml medicine bottle, and sufficient growth medium was added to moisten the explants. After incubation at 37~ for 1-2 days, most of the pieces were firmly adherent to tile glass, and further medium was added to cover the explants. Fibroblasts grew out from many of these pieces within 2 weeks, and by 4 weeks a monolayer of fibroblasts covered several square centimeters of the glass surface. This monolayer was harvested with a solution composed of 0.05 % trypsin and 0.2 ~ verseue in tris buffer at pH 7.4 and transferred to coverslips in Leighton tubes. Endothelial and fibroblast monolayers were usually examined after fixation in 10 % formalin in Ringer-Locke solution and staining with DELAIqELD'S hematoxylin and eosin. For special purposes Sudan IV, periodic acid-Schiff, and benzidine stains were used. In some specimens the intercellular "cement" was stained black with silver nitrate befole fixation. This was done by covering the coverslip successively with 0.4 ~ silver nitrate (2 minutes), 5 ~ glucose (3 changes), a solution containing 3 ~ cobalt bromide and 1 ~ ammonium bromide (1 minute), and finally, 5 ~o glucose (3 changes). In order to identify nuclei that were actively synthesizing DNA an autoradiographic technique was employed. The cell cultures were exposed for half an hour to culture medium containing 1/~C/ml of tritiated thymidine. The monolayers were then fixed in Ringer-Locke-formalin, dehydrated in absolute alcohol and allowed to dry for 12 hours. They were then coated with NTB-3 emulsion (Eastman Kodak), exposed in a light-tight box for 2 days, developed, and stained through the fixed emulsion with MACNEAL'S tetrachrome stain 1I. When this technique was used to compare endothelial cells with fibroblasts, the two cell types were subcultured onto fresh coverslips and treated with 3H thymidine and fixed at identical intervals after subculture. In some experiments pbagocytosis of carbon particles (India ink) by cells in culture was studied using a standard suspension made by adding 2 ml of Higgin's India ink to 100 ml of the growth medium. Phagocytosis was also studied by exposing the culture to growth medium containing human blood cells, chicken blood cells, sheep blood ceils, endothelial cells previously labelled with India ink, and heat-killed bacteria (Bacillus subtilis or Staphylococcus epidermidis). RESULTS
Venous endothelium Histologic sections of cords stained by Verhoeff's elastic stain showed that most of the endothelial cells had been detached from the trypsinized vein leaving the underlying internal elastic lamina intact. Sections of untrypsinized vein showed 51o cells lying between the endothelial Iayer and the internal elastic lamina (Figs. 1 and 2). When the cell suspensions were allowed to settle over a coverslip, the initially spherical cells began to flatten out on the glass surface within 3 hours. As the cells flattened they spread to cover a larger area, and by 12-24 hours the spreading process was complete. After this time any parts of the glass surface not covered by the cells remained uncovered. Although a slight shift in the position of individual cells occurred J. Atheroscler. Res., 6 (1966) 151-163
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after the first 24 hours, the m o n o l a y e r did n o t spread b y replication a n d m i g r a t i o n as was seen in fibroblast cultures. The flattened endothelial cells formed a sheet with each cell a b u t t i n g against the edge of neighboring cells. I n fixed p r e p a r a t i o n s there w~s often a narrow space between a d j a c e n t cells. The cells were polygonal or slightly elongated, a n d there was a r e m a r k a b l e u n i f o r m i t y in size of b o t h cell bodies a n d nuclei (Fig. 3). The cells were 40-50 # in their longest axis, a n d the nuclei 20 • 10/~ in their greatest a n d least diameters. The nucleus u s u a l l y c o n t a i n e d 1 or 2 nucleoli and very seldom more t h a n 3. Some cells c o n t a i n e d scattered sudanophilic droplets in the cytoplasm. The n u m b e r of cells with this appearance increased with the age of the culture. Silver s t a i n i n g gave black " c e m e n t lines" with endothelial cells grown either on collagen or on glass (Fig. 5). W i t h h e m a t o x y l i n staining m a n y cells showed dark, straight or slightly curved, parallel filaments (tonofibrils) in their cytoplasm. The m i t o c h o n d r i a were filamentous a n d were best seen b y phase c o n t r a s t microscopy. A few endothelial cultures were m a i n t a i n e d for 6 weeks, b u t more c o m m o n l y the cells became irregular in shape a n d detached from the s u b s t r a t u m after 2 - 5 weeks. I t was n o t possible to m a i n t a i n t h e m b y s u b c u l t u r e when glown in m e d i u m e~riched with either bovine fetal or h u m a n cord serum. W h e n cell counts were made before a n d after coverslip culture, no increase in cell n u m b e r was detected. Mitoses were occasionally seen b u t were e x t r e m e l y rare (Fig. 4). W h e n I n d i a ink suspension was added to the endothelial cultures, black granules appeared in their cytoplasm after a few hours a n d c o n t i n u e d to a c c u m u l a t e for several days (Fig. 6). Most of the granules congregated in the region m i d w a y b e t w e e n the nucleus and the edge of the cell leaving the i m m e d i a t e perinuclear a n d circumferential regions clear (Fig. 7).
Fig. 1. Cross section through wall of umbilical vein before trypsinization. The internal elastic lamina is clearly seen; and where it is most plicated, the endothelial cells assume columnar form. Verhoeff's elastic stain; • 188. Fig. 2. Cross section through wall of umbilical vein after trypsinization, Most of the endothelial cells have been detached, and the internal elastic lamina remains as a barrier preserving the integrity of subendothelial tissues. Verhoeff's elastic stain; • 406. Fig. 3. Umbilical vein endothelial cell culture monolayer. Cells are fairly uniform in shape and size. Many have cytoplasmic inclusions. Hematoxylin and eosin; • 188. Fig. 4. Mitotic figure in endothelial cell culture. Hematoxylin and eosin; • 714. Fig. 5. Endothelial cell culture on glass coverslip surface stained with silver nitrate and subsequently with hematoxylin and eosin. A black "cement line" surrounds many of the cells, x 714. Fig. 6. Endothelial culture after incubation for three hours with growth medium containing India ink suspension. A spindle-shaped "atypical" cell (macrophage) is identified by black granules in the cytoplasm. Hematoxylin and eosin; • 406. Fig. 7. A rounded macrophage in the same culture as shown in Fig. 6. The ink granules are concentrated near the nucleus in the center of the cell. Hematoxylin and eosin; x 406. Fig. 8. Same culture as Figs. 6 and 7 showing a macrophage with morphologie appearance intermediate between spindle-shaped and rounded forms. Hematoxylin and eosin; • 714. Fig. 9. "Typical" endothelial cells with black granules after 24 hours exposure to India ink. Hematoxylin and eosin; X 406. Fig. 10. Fibroblast culture after same exposure to India ink as the endothelial cells in Fig. 9. The cytoplasm contains few ink granules. Hematoxylin and eosin; • 406.
j . Atheroscler. Res., 6 (1966) 151-163
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j . Atheroscler. Res., 6 (1966) 151-163
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D.G. FRYER, G. BIRNBAUM, C. N. LUTTRELL There were a n u m b e r of " a t y p i c a l " cells wich t o o k up the ink v e r y r a p i d l y a n d
within 3 - 6 hours b e c a m e intensely black (Figs. 9 and 10). Many d e v e l o p e d large, clear vacuoles and b e c a m e d e t a c h e d from the coverslip. This d a m a g e could be p r e v e n t e d by limiting the t i m e of exposure to India ink to 3 hours. The " a t y p i c a l " cells were either circular or were elongated a n d spindle-shaped, often with e x p a n d e d fan-like bulbs at one or b o t h ends of the cell. The nuclei of these cells were usually slightly smaller and stained more densely t h a n the " t y p i c a l " cells, and the c y t o p l a s m had a reticular appearance. W i t h Sudan I V staining most of these cells appeared loaded w i t h fat droplets and were t h e r e b y c o n t r a s t e d with the " t y p i c a l " cells. The i d e n t i t y of the circular and e l o n g a t e d varieties of " a t y p i c a l " cells was suggested b y the presence of i n t e r m e d i a t e forms (Fig. 8) t h a t h a d all the characteristics of a circular celI b u t had a single slender process of c y t o p l a s m e x t e n d i n g from one side of the cell. In p a t c h y m o n o l a y e r s " a t y p i c a l " cells were often found in isolation some distance a p a r t from the confluent endothelial cell patches. W h e n elongated " a t y p i c a l " cells were found a m o n g " t y p i c a l " cells, t h e y a p p e a r e d to lie o v e r t h e m r a t h e r t h a n p a r t i c i p a t e in the f o r m a t i o n of the continuous sheet of cells. W h e n cultures were e x a m i n e d on a w a r m stage w i t h the phase c o n t r a s t microscope, the " a t y p i c a l " cells were seen to show c o n s t a n t changes in c o n t o u r b y the c o n t i n u a l protrusion and r e t r a c t i o n of pseudopodia. I n cultures 3 or 4 weeks old the cell bodies of m a n y of these cells were g r e a t l y enlarged, up to 200 # in diameter, a l t h o u g h the nucleus showed only a slight increase in size.
Fig. 11. This specimen was produced by labelling an endothelial culture with India ink, as shown in Fig. 9. The labelled culture was trypsinized, and the separated cells suspended in culture medium covering an unlabelled monolayer of endothelial cells. After incubation for 24 hours, many inklabelled cells were found as inclusions containing black granules. Hematoxylin and eosin; • 714. Fig. 12. Inclusions in endothelial cells after 12 hours incubation in growth medium containing human red cells. Hematoxylin and eosin; • 714. Fig. 13. Endothelial cell culture after 48 hours incubation with growth medium containing human red cells. Some endothelial cells have as many as ten included erythrocytes. Hematoxylin and eosin; • 406. Fig. 14. Endothelial cell culture after 6 hours incubation in a suspension of heat-killed B. subtilis. Hematoxylin and eosin; • 714. Fig. 15. Endothelial culture cells after incubation for 3 hours in growth medium containing heatkilled Staph. epidermidis. A macrophage is seen loaded with staphylococci. Hematoxylin and eosin; • 714. Fig. 16. Culture exposed successively to 3H thymidine for half an hour and India ink suspension for 3 hours. A macrophage is shown labelled by both aH thymidine and ink. Culture stained with 3JiACNEAL'S tetraehrome through the photographic emulsion; • 714. Fig. 17. Umbilical cord fibroblast culture in which several nuclei are labelled with ZH thymidine. Cells have overlapping and anastomosing processes that form a reticulum. Unlabelled nuclei have several darkly staining nucleoli. ~[AC~X~EAL'Stetrachrome; • 714. Fig. 18. Endothelial cell culture obtained by trypsinization of umbilical artery. Culture was exposed to India ink for 3 hours and fixed at 5 days. A small macrophage labelled with ink and a large fibroblast are shown. Hematoxylin and eosin; • 188. Fig. 19. Ten day old specimen of the same culture shown in Fig. 18. Only fibroblasts are clearly recognizable. Hematoxylin and eosin; • 188. Fig. 20. Four week old umbilical vein endothelial culture showing a very large macrophage. HematoxyIin and eosin; • 188. J. Atheroscler. Res., 6 (1966) 151-163
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In caltures one week old about 20 ceils per 1000 wele of this "'atypical" form, but the proportion showed a slight steady increase, and in 4-week cultures there were usually about 64 per 1000. When these cells were labelled with India ink, it was found that further "atypical" cells were not formed by transformation of "typical" cells. After exposure of cultures of 3H thymidine for half an hour, only 6-9 endothelial cell nuclei per 1000 were labelled. Occasional "atypical" cells were also labelled but it was not possible to show a difference in the frequency of labelling of "typical" and "atypical" cells. In old cultures in which some cells had begun to assume irregular shapes, no labelling was found after similar exposure to 3H thymidine. Many endothelial cells in culture contained large, weakly eosinophilic, PASpositive, cytoplasmic inclusions. Often these inclusions were surrounded by a nalrow clear zone, and frequently the nucleus was indented or pushed to one side by an inclusion. When living cultures were examined by phase contrast microscopy, it was observed that not all the initially spherical cells flattened out. A few remained spherical, and when they lay in close iuxtaposition to flattened cells, they were seen in subsequently stained preparations to have become eosinophilic inclusions. In some experiments endothelial cultures were exposed to India ink until all the cells contained numerous black granules. These cells were then trypsinized and the separated cells suspended in growth medium which was added to endothelial monolayers. Inclusions containing numeroos black granules were subsequently demonstrated in the cells of these monolayers (Fig. 11). Human, chicken and sheep erythrocytes were also phagocytosed by endothelial cultures. Most of the endothelial cells acquired from 1-10 included erythrocytes within 48 hours (Figs. 12 and 13). The included blood cells were at first recognizable by their shape and size and by staining with Ralph's benzidine stain. Within a day the inclusions became fragmented and lost the staining characteristic of erythrocytes. Killed Bacillus subtilis and Staphylococcus epidermidis suspended in the growth medium were also phagocytosed (Figs. 14 and 15). "Atypical" cells showed a much greater affinity for India ink and staphylococci than "typical" cells (Fig. 16), but there was no apparent difference between the two cell types in regard to their propensity to ingest the Bacillus subtilis or cellular particles.
Arterial endothelium Histologic sections of umbilical cold showed no distinct internal elastic lamina in the mnbilical artery, and after trypsinization of the artery there was some fragmentatioll of the subendothelial tissue. The cell cultures initially differed from those derived from vein only by the presence of cells which had all the characteristics of fibroblasts. At first these fibroblasts were few in number, but within 2 weeks these cultures could be distinguished from pure fibroblast cultures only by the presence of occasional "atypical" cells that retained India ink (Fig. 18). The orderly arrangement of the endothelial cells was completely lost in the overgrowth of fibroblasts, and "typical" endothelial cells could not be recognized with certainty (Fig. 19). J. Atheroscler. Res., 6 (1966) 151-163
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Fibroblasts
No differences were detected between fibroblasts of umbilical cord and prepuce origins. Fibroblasts were irregular in shape, often greatly elongated, and sent out processes that overlapped adjacent cells forming a network with the processes of other cells. They varied greatly in size, and in large cells the nucleus often measured 50/2 in its greatest diameter. In general the nucleoli were more numerous and stained more intensely than those of endothelial cells (Fig. 17). Silver staining showed a black "cement line" similar to that seen in endothelial cultures. Tile larger fibroblasts showed darkly staining parallel filaments (tonofibrils) in their cytoplasm. Unlike endothelial cultures, fibroblasts replicated rapidly and could be readily subcultured and maintained for several months. In experiments with taitiated thymidine fibroblast nuclei took up thymidine more frequently than endothelial nuclei, and cell counts showed that 50-70 nuclei per 1000 were labelled after 30 minutes exposure to aH thymidine. Fibroblast cultures showed a very slight capacity for phagocytosis of India ink and cellular and bacterial particles.
DISCUSSION The nature and origin of a ceil cannot be determined with certainty by its morphology in culture. Since the vein internal elastic lamina was not damaged there is no doubt that the cells in the cultures derived from veins were those which lie superficial to the elastic lamina. It is on this basis, rather than morphology, that we are confident that our cultures were comprised predominantly of endothelial cells. Although endothelial cells certainly predominated, other cells may have been included such as cells which m a y occasionally lie between the elastic lamina and the endothelium, and blood cells which had become firmly attached to the endothelium. Despite these uncertainties our study sheds some light on the nature of endothelial cells, especially their relationship to fibroblasts and macrophages. There is a long-standing controversy about the relationship of endothelial cells to fibroblasts. MAXlMOWlz, in 1925, stated that in tissue culture endothelial cells grew out of the end of an explanted piece of blood vessel but soon could not be distinguished from fibroblasts; the 1957 edition of MAXlMOW AND BLOOM'S textbook 13 still maintains that endothelial cells in the body can become changed into fibroblasts. Moreover, ALTSCttUL14 in an extensive review of the literature concluded: " I t is difficult ot impossible to distinguish in tissue culture between endothelial cells and fibroblasts. Furthermore, there is the rather common belief that endothelial cells in tissue culture will turn into fibroblasts or fibroblast-like cells." However, KHLOPIN AND CHISTOVA7 have more recently stated: "rLeither in tissue culture nor in repair regeneration is endothelium ever converted into connective tissue". Pure culture of human endothelial celts from normal blood vessels has been j . Atheroscler. Res., 6 (1966) 151-163
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reported previously only by MARUYAMA9. MARUYAMAused umbilical vein as his source of ceils but was not successful in culturing fibroblasts from umbilical cord for comparison with the endothelial cells. In our investigations fibroblasts were grown from umbilical cord and from human foreskin, and comparison with endothelial cells was, therefore, possible. The fibroblasts grown from cord and foreskin were identical in appearance and behaviour and corresponded to the description of fibroblasts (mechanocytes) in culture given by WII-LMER15. MARUYAMA9 found that the intercellular "cement" stained black with silver nitrate in endothelial cultures grown on collagen but not in those grown on glass. He considered the presence of this cement line to be evidence for the endothelial origin of the ceils, tn our own experience the cement line stained equally well in cultures grown on glass and on collagen. However, we do not think these lines are especially significant as they were found also in our fibroblast cultures. ALTSCHUL14 also pointed out that this staining property cannot be considered as specifc for a single cell type. MAXlMOW1~ attached importance to the presence of tonofibrils in identifying fibroblasts. However, MARUYAMA9 iound similar "tension striae" in endothelial cells, and we also observed this appearance in both fibroblasts and endothelial cells. Nevertheless, there were definite morphological and behaviourat differences that clearly distinguished fibroblasts from endothelial cells. "Typical" endothelial cells showed little variation in size, and they spread out to form sheets; their nucleoli stained faintly and were few in number. By contrast, fibroblasts varied greatly in size, their processes overlapped and formed networks; their nucleoli were numerous and stained intensely. Further points of distinction were the greater capacity for phagocytosis in endothelial cells, and the greater rate of replication and DNA synthesis in fibroblasts as shown by the greater number of nuclei labelled with aH thymidinelT. The "atypical" endothelial cells which we noted in small numbers in our cultures of endothelium resembled fibroblasts in being spindle-shaped, and in older cultures their cell bodies grew to a size achieved b y the largest fibroblasts. However, unlike fibroblasts, they did not replicate so fast as to overgrow the "typical" endothelial cells, they did not form a reticulum of branching cells, and they had a distinctively high affinity for India ink. In disintegrating endothelial cultures the cells were often irregular in outline and elongated. These cells showed no evidence of DNA synthesis, and there is no reason to believe that they represent a conversion of either "typical" or "atypical" endothelial cells to fibroblasts. The existence of more than one cell type in endothelial cultures has been described several times. Recently KOmE et al. 18, described "fibroblasts" and another cell which " m a y be an endothelial cell". The latter cell was rounded or oval and showed ameboid movements. These authors obtained their cultules by explanting whole thickness rabbit aorta and most probably subintimal cells were present in the cultures. SH1BUYA 6, in 1931, cultured explants of rabbit endothelium. He made the following statement: "There developed after 5-6 days around the growth of the 'typical' endoJ. Athero~cler. Res., 6 (1966) 151-163
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thelial cells, some which were different from the characteristic cells. They had spindlelike or roundish bodies". This description is very applicable to our "atypical" cells. MARUYAMA9 stated: "Infrequently some cells transformed into fibroblast-like elements. For example, in certain series of cultures made from an umbilical vein, there were no fibroblast-like cells after 2-4 days in culture, but they appeared after 10-12 days. These cells were spindle-shaped with slender processes". Again, this description fits our "atypical" cells, but neither SHIBUYA6 nor MARUYAMA9 described how the spindle-shaped cells reacted with India ink. KHLOPIN AND CHISTOVA7 cultured cells from explants of rabbit vena cava endothelium and described the appearance of the culture when India ink was added. They found: "After several days the amount of India ink taken up b y the cells increases, but remains unevenly distributed between the various cells of the growth zone. Some of them contain either few granules or none, while in others there m a y be a considerable number. Gradually, the cells which are crowded in India ink granules become rounded off and disorganized". PTOKHOV19 grew endothelial cultures from explants of rabbit blood vessels and studied the phagocytic properties of cells derived in this way. Some cells became overloaded with absorbed particles and became isolated and rounded into "ball forms" with the appearance of macrophages. LAZZARINI5 described two types of cells derived from human aortic intima, one type showing a rapid uptake of colloidal carbon and in certain circumstances becoming "ring t y p e " ceils. We cannot say whether there is a resemblance between endothelial cells obtained from human umbilical artery and vein and those derived from rabbit and human adult blood vessels. The descriptions of the rounded and ameboid "endothelial" cells of KOlDE et al. Is, the "spindle-shaped and iounded cells" of GHIBUYA 6, the "fibroblast-like elements" of MAREYAMA 9, the cells "crowded with India ink" of KtfLOPIN AND CHISTOVA 7, the "isolated and rounded" cells of PTOKHOV19and the "ring type" cells of LAZZARINI5 suggest some resemblances to tile "atypical" cells we have described, but we do not presume that they are analogous. What, then, is the significance of the two distinct endothelial cell types which we have designated as "typical" and "atypical"? We found that both cell types were recognizable as soon as the culture had flattened out. If the atypical cells were damaged by prolonged exposure to India ink, they weIe not replenished by transformation of the remaining "typical" cells. The greater abundance of these cells in older cultures m a y be explained by their more rapid replication. WILLMER20 stated that in general in tissue culture, cells change morphologically to one of three types which he called mechanocytes (fibroblasts), epitheliocytes and amebocytes. Mechanocytes have a reticular arrangement, while epitneliocytes are characterized by a tendency to adhere to each other and to coverslip surfaces and to spread in a uniform sheet with each cell firmly attached to its next-door neighbors by a cement substance. Amebocytes remain detached from one another, are strongly phagocytic, store carbon particles and show ameboid motion. Using these criteria, we would classify the "typical" cells as epitheliocytes (in WILLMER'S classification) and the "atypical" as amebocytes; however, both cell types have a remarkable ability to phagocytose cellular debris. J. Atheroscler. Res., 6 (1966) 151-163
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D. G, F R Y E R , G. BIRNBAUM, C. N. LUTTRELL
Our cultures were composed mostly of "typical" cells which we, thelefore, regard as endothelial cells proper. WlLLMER~0 points out that in vitro monocytes and histiocytes (macrophages) behave as amebocytes, and we think that the "atypical" cells we have described are most probably macrophages that exist in close association with endothelium. I t is well established that macrophages can be cultured from blood lymphocytes and monocytes16, 21-2a, and indeed, we have cultured cells with the appearance of "atypical" cells from cord blood. However, macrophages ("atypical" cells) appeared in our cultures in the absence of erythrocytes and were, therefore, not derived from contaminating blood. We cannot exclude the possibility that lymphocytes or monocytes had become attached to the endothelium either during fetal life or after the placental circulation ceased. The conversion c)f blood macrophages to fibroblast-like cells has been described16, zs, although other authors have not observed this change even after prolonged culture of blood macrophages 22. In our cultures some macrophages became very large, but in appearance and behaviour they remained distinct from fibroblasts. The use of trypsin m a y produce chromosomal aberrations that alter metabolism and cell growth 24. However, there is no evidence for this change in short-term cultures of the kind we gave studied, and we do Itot think that the cell differences we have seen could have been caused artifactually b y trypsin treatment. CONCLUSIONS
Normal human endothelial cells derived from umbilical vein m a y be grown in cell cultule. Endothelial cell cultures derived by tr2r of umbilical arteries were initially similar to those from the vein, but a small number of fibroblasts contaminated the cultures and replicated so rapidly that the culture soon resembled a culture of pure fibroblasts. This contamination of arterial endothelial cultures by fibroblasts was associated with the absence of a distinct ivternal elastic lamina in this artery. In cultures free of fibroblasts two distinct cell types were found. Both types could be distinguished morphologically and behaviourally from fibroblasts and neither type was converted into fibroblasts. The more numerous cell type was considered to be a true endothelial cell. The less numerous ceils were thought to be macrophages but it was not determined whether they were derived Irom intimal celIs or from blood cells that adhered to endothelium. Macrophages were not formed b y metamorphosis of ordinary endothelial cells. Both cell types had a very marked ability to phagocytose cellular and bacterial particles, macrophages usually, but not always having gleater phagocytic capacity depending on the nature of the palticle being ingested. ACKNOWLEDGEMENTS
The authors are grateful to Miss NANCY BUNKER for expert technical assistance. The funds were made available for this research from the University of Washington, General Research Support Grant, P H S number F R 05132-01. j . Atherosder. Res., 6 (1966) 151-163
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SUMMARY
H u m a n endothelial cells d e r i v e d b y t r y p s i n i z a t i o n of umbilical v e i n a n d a r t e r y were grown in cell c u l tu r e a n d c o m p a r e d w i t h fibroblasts g r o w n from e x p l a n t s of umbilical cord. Tw o t y p e s of cells were recognized in endothelial cultures: endothelial cells p r o p e r an d macrophages. T h e f o r m e r replicated v e r y slowly and could n o t be m a i n t a i n e d b y s u b c u l t m e . Macrophages c o n s t i t u t e d a b o u t 5 % of the t o t a l cell population. T h e y r e p l i c a t e d slightly more r a p i d l y t h a n t h e true en d o t h el i al cells a n d were n o t d e r i v e d from these cells. N e i t h e r cell t y p e was t r a n s f o r m e d into fibroblasts. B o t h cell t y p e s h a d m a r k e d ability to p h a g o c y t o s e cellular a n d b a c t e r i a l particles, mac~ophages sometimes, b u t n o t always, h a v i n g g r e a t e r p h a g o c y t i c c a p a c i t y d ep en d i n g on the n a t u r e of t h e particle ingested. E n d o t h e l i a l cultures derived from umbilical a r t e r y initially differed from those from the vein only b y t h e presence of a small n u m b e r of fibroblasts which, in older cultures, overgrew the en d o t h el i al cells. Th e presence of fibroblasts in arterial cultures was associated w i t h t h e absence of a distinct i u t e l n a l elastic l a m i n a so t h a t s u b e n d o t h e l i a l cells were included in the culture. REFERENCES 1 M. R. MURRAYAND A. P. STOUT, Am. J. Pathol., 20 (1944) 277. 2 G. CAMERONAND R. CHAMBERS,Am. J. Cancer, 30 (1937) 115. 8 D. R. COMAN, Cancer Res., 2 (1942) 618. 4 W . C. WOODARD AND C. M. POMERAT, Anat. Rec., 117 (1953) 663. 5 A. A. LAZZARINI,Doctoral Thesis, Cornell University, 1959. e T. SHIBUYA, Kitasato Arch. Exptl. Med., 8 (1931) 68. 7 N. G. KHLOPIN AND N. M. CHISTOVA,Dokl. Akad. Nauk S S S R , 119 (1958) 217. 8 C. M. POMERATAND W. C. SLICK, Nature, 198 (1963) 859. 9 y. MARUYAMA,Z. Zellforsch., 60 (1963) 69. 10 R . L . EHRMANN AND G. O. GEY, J. Natl. Cancer Inst., 16 (1956) 1375. Xl W. J. MACNEAL,J. Am. Med. Assoc., 78 (1922) 1122. 12 A. A. MAXIMOW,Anat. Rec., 29 (1925) 369. 18 A. A. MAXIMOWAND W. BLOOM, A Textbook of Histology, 7th edition, W. B. Saunders Co., Philadelphia, 1957, p. 47. 14 R. ALTSCHUL,Endothelium, Its Development, Morphology, Function and Pathology, MacMillan Co., New York, 1954, pp. 10, 37. 15 E. N. WILLMER, Tissue Culture. The Growth and Differentiation of Normal Tissues in Artificial Media, 3rd edition, Methuen and Co., Ltd., London, 1958, p. 9. 16 A. A. MAXlMOW,Arch. Exptl. Zellforsch., 5 (1928) 169. 17 C. P. LEBLOND, B. MESSIER AND B. KOPRIWA, Lab. Invest., 8 (1959) 296. 18 R. KOIDE, O. J. POLLACKAND D. A. BURNS, J. Atheroscler. Res., 3 (1963) 32. 10 M. P. PTOKHOV, Arch. Anat. Histol. Embryol. (Leningrad), 8 (1963) 75. 20 E. N. WILLMER, Essays on Growth and Form, Oxtord University Press, Oxford, 1945, pp. 265, 272. 21 M. R. LEWIS, Am. J. Pathol., 2 (1925) 91. e2 L . BERMAN AND C. S. STULBERG, Lab. Invest., 11 (1962) 1322. 28 W. BLOOM, Handbook of Hematology, Hoeber, Inc., New York, 1938, Sec. XX, p. 1507. 24 G. BARSKI AND R . CASSINGENA, J. Natl. Cancer Inst., 30 (1963) 865. J. Atheroscler. Res., 6 (1966) 151-163